Administration of nedd8-activating enzyme inhibitor and hypomethylating agent

ABSTRACT

The present disclosure relates to methods for the treatment of cancer in patients in recognized need of such treatment. The methods comprise administering to such a patient an NAE inhibitor or a pharmaceutically acceptable salt thereof, such as ((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl sulfamate (MLN4924) or {(1S,2S,4R)-4-[(6-{[(1R,2S)-5-chloro-2-methoxy-2,3-dihydro-1H-inden-1-yl]amino}pyrimidin-4-yl)oxy]-2-hydroxycyclopentyl)methyl sulfamate (I-216), and a hypomethylating agent or a pharmaceutically acceptable salt thereof, such as azacitidine or decitabine. Also disclosed are medicaments for use in the treatment of cancer.

This application claims benefit of priority from U.S. Provisional PatentApplication No. 61/555,049 filed on Nov. 3, 2011.

Inhibition of NEDD8-activating enzyme (NAE) has been shown to inducecancer cell death and inhibit the growth of tumors in xenograft models.See, e.g., T. A. Soucy et al., Nature, 2009, 458, 732-737; T. A. Soucyet al., Clin. Cancer Res., 2009, 15 (12), 3912-3916; and J. E. Brownellet al., Mol. Cell., 2010, 37 (1), 102-111. Reports of Phase I clinicalstudies of an NAE inhibitor include R. T. Swords et al., Blood, 2010,115, 3796-3800; J. S. Kauh et al., J. Clin. Oncol., 2011, 29, abstract3013; and S. Bhatia et al., J. Clin. Oncol., 2011, 29, abstract 8529.Inhibitors of NAE are described in U.S. patent application Ser. No.11/346,469 (Publ. No. 2006/0189636, U.S. Pat. No. 7,951,810), Ser. No.11/700,614 (Publ. No. 2007/0191293) and Ser. No. 11/890,338 (Publ. No.2008/0051404, U.S. Pat. No. 8,008,307), each of the aforementionedpublications is hereby incorporated by reference herein in its entirety.If there is any discrepancy between any of these documents and thepresent specification, the present specification controls.

Hypomethylatlng agents have been approved by the US Food and DrugAdministration in the treatment of cancer. For example, VIDAZA®(azacitidine for injection) is indicated for treatment of patients withthe following French-American-British (FAB) myelodysplastic syndromessubtypes: refractory anemia (RA) or refractory anemia with ringedsideroblasts (if accompanied by neutropenia or thrombocytopenia orrequiring transfusions), refractory anemia with excess blasts (RAEB),refractory anemia with excess blasts in transformation (RAEB-T), andchronic myelomonocytic leukemia (CMMoL). DACOGEN® (decitabine forinjection) is indicated for treatment of patients with myelodysplasticsyndromes (MDS) including previously treated and untreated, de novo andsecondary MDS of all French-American-British subtypes (refractoryanemia, refractory anemia with ringed sideroblasts, refractory anemiawith excess blasts, refractory anemia with excess blasts intransformation, and chronic myelomonocytic leukemia) and intermediate-1,Intermediate-2, and high-risk International Prognostic Scoring Systemgroups.

The highest possible dose (MTD: maximum tolerated dose) is typicallysought for agents for the treatment of cancer because the benefit of thetreatment is believed to increase with dose. See, e.g., Y. Lin and W. J.Shih, Biostatistics, 2001, 2 (2), 203-215. A synergistic combination ofagents—that is, a combination of agents that is more effective than isexpected from the effectiveness of its constituents, without alsocompounding the treatment side effects—can provide an opportunity todeliver even greater efficacy at the MTD. Accordingly, it can bedesirable to discover synergistic combinations of anti-cancer agents inorder to treat cancer patients most effectively, without overloading thepatient with side effects.

It has now been discovered that the administration of an NAE inhibitoror a pharmaceutically acceptable salt thereof and a hypomethylatingagent or a pharmaceutically acceptable salt thereof provides asynergistic effect. Both in vitro and in vivo synergistic effects werefound. In vitro synergy was measured using The Combination Index (M. C.Berenbaum, J. Theor. Biol., 1985, 114, 413-431), as discussed in furtherdetail below. In vivo synergy was measured according to a synergysurvival method or a synergy tumor growth method, as discussed infurther detail below.

At least one aspect of the present disclosure relates to methods oftreating cancer comprising administering to a patient in need of suchtreatment, a therapeutically effective total amount of an NAE inhibitoror a pharmaceutically acceptable salt thereof and a hypomethylatingagent or a pharmaceutically acceptable salt thereof to a subject in needof such treatment.

At least one aspect of the present disclosure is also directed towardsthe use of an NAE inhibitor or a pharmaceutically acceptable saltthereof with a hypomethylating agent or a pharmaceutically acceptablesalt thereof for treating cancer in a patient in need of such treatment.

At least one aspect of the present disclosure relates to a kitcomprising at least one medicament for use in treating cancer in asubject in recognized need thereof. For example, the kit may comprise atleast one medicament comprising at least one dose of an NAE inhibitor ora pharmaceutically acceptable salt thereof, and instructions foradministering the at least one medicament with a hypomethylating agentor a pharmaceutically acceptable salt thereof, or the kit may compriseat least one medicament comprising at least one dose of ahypomethylating agent or a pharmaceutically acceptable salt thereof, andinstructions for administering the medicament with an NAE inhibitor or apharmaceutically acceptable salt thereof. In various embodiments, thekit can comprise at least one medicament comprising at least one dose ofan NAE inhibitor or a pharmaceutically acceptable salt thereof and atleast one medicament comprising at least one dose of a hypomethylatingagent or a pharmaceutically acceptable salt thereof, and instructionsfor administering the medicaments. Furthermore, for example, the kit cancomprise anti-cancer actives consisting of at least one medicamentcomprising at least one dose of an NAE inhibitor or a pharmaceuticallyacceptable salt thereof, and at least one medicament comprising at leastone dose of a hypomethylatlng agent or a pharmaceutically acceptablesalt thereof; said kit for treating cancer further comprising dosinginstructions for administering the medicaments for treatment of thesubject in recognized need thereof.

At least one aspect of the present disclosure relates to at least onemedicament for use in treating cancer in a subject in need of suchtreatment. For example, the at least one medicament may comprise an NAEinhibitor or a pharmaceutically acceptable salt thereof, or ahypomethylating agent or a pharmaceutically acceptable salt thereof, ora combination thereof.

At least one aspect of the present disclosure relates to the use of anNAE Inhibitor or a pharmaceutically acceptable salt thereof in themanufacture of at least one medicament for treating cancer, wherein theNAE inhibitor or a pharmaceutically acceptable salt thereof isadministered with a hypomethylating agent or a pharmaceuticallyacceptable salt thereof to a patient in need of such treatment.

At least one aspect of the present disclosure relates to the use of ahypomethylating agent or a pharmaceutically acceptable salt thereof inthe manufacture of at least one medicament for treating cancer, whereinthe a hypomethylating agent or a pharmaceutically acceptable saltthereof is administered with an NAE inhibitor or a pharmaceuticallyacceptable salt thereof to a patient in need of such treatment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the Combination index values for NAE inhibitors MLN4924 andI-216, each separately in combination with azacitidine or decitabine inHL60, OCIM2, NB4 and THP-1 cell lines.

FIG. 2 shows a plot of tumor volume as a function of time in an HL-60subcutaneous xenograft model following subcutaneous treatment with: thevehicle alone, MLN4924 as a single agent, azacitidine (“Aza”) as asingle agent, and co-administration (s.c.) of MLN4924 and azacitidine,on Days 1, 4, 8, 11, 15 and 18 at the indicated doses.

FIG. 3 shows a plot of tumor volume as a function of time in a THP-1subcutaneous xenograft model following subcutaneous treatment with: thevehicle alone, MLN4924 as a single agent, azacitidine (“Aza”) as asingle agent, and co-administration (s.c.) of MLN4924 and azacitidine,on Days 1, 4, 8, 11, 15 and 18 at the Indicated doses.

FIG. 4 shows a plot of tumor volume as a function of time in a OCI-M2subcutaneous xenograft model following subcutaneous treatment with: thevehicle alone, MLN4924 as a single agent, azacitidine (“Aza”) as asingle agent, and co-administration (s.c.) of MLN4924 and azacitidine,on Days 1, 4, 8, 11, 15 and 18 at the indicated doses.

FIG. 5 shows a plot of percentage survival as a function of time in anHL60 disseminated model following subcutaneous treatment with: thevehicle alone, MLN4924 as a single agent, azacitidine (“AzaC”) as asingle agent, and co-administration (s.c.) of MLN4924 and azacitidine onDays 22, 25, 29, 32, 36, 39 at the indicated doses.

The following definitions and abbreviations may be used herein:

-   ALP alkaline phosphatase-   ALT alanine aminotransferase-   AML acute myelogenous leukemia-   ANC absolute neutrophil count-   AST aspartate aminotransferase-   AUC area under the plasma concentration versus time curve-   BSA body surface area-   CR complete response-   CRM continual reassessment method-   CYP cytochrome P450-   DLBCL diffuse large B-cell lymphoma-   DLT dose-limiting toxicity-   LFT liver function tests-   LVEF left ventricular ejection fraction-   MDS myelodysplastic syndromes-   MM multiple myeloma-   MTD maximum tolerated dose-   NAE Nedd8-activating enzyme-   NEDD8 neural precursor cell expressed, developmentally    down-regulated 8-   PASP pulmonary artery systolic pressure-   PR partial response-   QD once daily-   SCLC small cell lung cancer

As used herein, “dose-limiting toxicity” (DLT) is defined as a negativeevent considered by the administering physician to be related to therapywith MLN4924 such that the administering physician believes the dosesshould be limited in quantity or altogether stopped. Examples of suchevents include:

-   -   Grade 4 neutropenia (ANC <500 cells/mm³) lasting more than 7        consecutive days    -   Grade 3 neutropenia with fever and/or infection, where fever is        defined as an oral temperature ≧38.5° C.    -   Grade 4 thrombocytopenia (platelets <25,000/mm³ but >10,000/mm³)        lasting more than 7 consecutive days    -   Grade 3 thrombocytopenia with bleeding    -   A platelet count <10,000/mm³ at any time    -   Grade 3 or greater nausea and/or emesis despite the use of        optimal anti-emetic prophylaxis (wherein “optimal anti-emetic        prophylaxis” is defined as an anti-emetic regimen that employs a        5-HT₃ antagonist given in standard doses and according to        standard schedules). Dexamethasone should not be used because of        its CYP3A-inducing effects.    -   Grade 3 or greater diarrhea that occurs despite maximal        supportive therapy    -   An absolute reduction in LVEF of ≦10% to a value <50% (e.g.,        LVEF=45% in a patient with LVEF=55% at baseline)    -   A decrease in LVEF to <40%    -   An increase in PASP to >50 mm Hg or 3× baseline    -   Any other Grade 3 or greater nonhematologic toxicity with the        following exceptions:        -   Grade 3 arthralgia/myalgia        -   Brief (<1 week) Grade 3 fatigue        -   Grade 3 fever that occurs in the absence of Grade 3 or worse            neutropenia or documented infection following daily            administration of MLN4924    -   Treatment delay of more than 1 week because of a lack of        adequate recovery of MLN4924-related hematological or        nonhematologic toxicities    -   MLN4924-related toxicity that requires that any doses of MLN4924        are missed during a cycle or discontinuation of therapy with        MLN4924

As used herein, “clinically effective amount” and “therapeuticallyeffective” means an amount of a therapeutic substance that is sufficientupon appropriate administration over an appropriate period of time to apatient (a) to cause a detectable decrease in the severity of thedisorder or disease state being treated; (b) to ameliorate or alleviatethe patient's symptoms of the disease or disorder; or (c) to slow orprevent advancement of, or otherwise stabilize or prolong stabilizationof, the disorder or disease state being treated (for instance, toprevent additional tumor growth or inhibit the cell growth of a cancer).

When more than one therapeutic substance is being administered, the“clinically effective total amount” or “therapeutically effective totalamount” means that the sum of the individual amounts of each therapeuticsubstance meets the definition of “clinically effective amount” even ifthe individual amounts of any number of the individual therapeuticsubstances would not. For example, if 10 mg of A were not a clinicallyeffective amount, and 20 mg of B were not a clinically effective amount,but the administration of 10 mg A+20 mg B resulted in at least one ofthe results enumerated for the definition of “clinically effectiveamount,” then the sum of 10 mg A+20 mg B would be considered a“clinically effective total amount.”

In any form or composition, the administered dose(s) or the clinicallyeffective (total) amount can be expressed as amount(s) of therapeuticsubstance(s) per patient BSA, e.g., as mg/me.

As used herein, “patient” means a human being diagnosed with, exhibitingsymptoms of or otherwise believed to be afflicted with a disease,disorder or condition and thus in recognized need of the treatmentdescribed herein.

As used herein, the illustrative terms “include,” “such as,” “forexample” and the like (and variations thereof, e.g., “includes” and“including,” “examples”), unless otherwise specified, are intended to benon-limiting. That is, unless explicitly stated otherwise, such termsare intended to imply “but not limited to,” e.g., “including” meansincluding but not limited to.

As used herein, “body surface area” (BSA) is calculated using a standardnomogram, e.g.,

${{BSA}\left( m^{2} \right)} = {{\sqrt{\frac{{Ht}\mspace{14mu} ({cm}) \times {Wt}\mspace{14mu} ({kg})}{3600}}\mspace{14mu} {or}\mspace{14mu} {BSA}} = \sqrt{\frac{{Ht}\mspace{14mu} ({cm}) \times {Wt}\mspace{14mu} ({kg})}{3600}}}$

Therapeutic Substances—NAE inhibitors.

The compound((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]-pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate:

also known as MLN4924, has been reported to be an inhibitor ofNEDD8-activating enzyme (NAE). See, e.g., T. A. Soucy et al., Nature,2009, 458, 732-737; T. A. Soucy et al., Clin. Cancer Res., 2009, 15(12), 3912-3916; and J. E. Brownell et al., Mol. Cell., 2010, 37 (1),102-111. As discussed above, MLN4924, pharmaceutically acceptable saltsthereof, pharmaceutical compositions of MLN4924 or a pharmaceuticallyacceptable salt thereof, processes for synthesis, and polymorphic formsthereof have been described previously. See, e.g., U.S. patentapplication Ser. No. 11/700,614 (Publ. No. 2007/0191293), Ser. No.12/221,399 (Publ. No. 2009/0036678) and Ser. No. 12/779,331 (Publ. No.2011/0021544). MLN4924 Drug Substance (“MLN4924-DS”) is thehydrochloride salt of MLN4924, i.e.,((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate hydrochloride.

The compound{(1S,2S,4R)-4-[(6-{[(1R,2S)-5-chloro-2-methoxy-2,3-dihydro-1H-inden-1-yl]amino}pyrimidin-4-yl)oxy]-2-hydroxycyclopentyl}methylsulfamate:

also known as 1-216, has also been reported to be an inhibitor of NAE.See U.S. patent application Ser. No. 13/592,389, filed Aug. 23, 2012,claiming priority to U.S. Provisional Patent Appl. No. 61/526,830, filedAug. 24, 2011, which are hereby incorporated by reference herein intheir entirety. If there is any discrepancy between these documents andthe present specification, the present specification controls.

Therapeutic Substances—Hypomethylating Agents.

Azacitidine is 4-amino-1-β-D-ribofuranosyl-s-triazin-2(1H)-one (IUPACname: 4-amino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one):

As discussed above, VIDAZA® (azacitidine for injection, CelgeneCorporation (Summit, N.J.); VIDAZA® is a registered trademark of CelgeneCorporation) is indicated and approved by the US FDA for treatment ofpatients with the following French-American-British (FAB)myelodysplastic syndromes subtypes: refractory anemia (RA) or refractoryanemia with ringed sideroblasts (if accompanied by neutropenia orthrombocytopenia or requiring transfusions), refractory anemia withexcess blasts (RAEB), refractory anemia with excess blasts intransformation (RAEB-T), and chronic myelomonocytic leukemia (CMMoL).Full prescribing Information for VIDAZA® is available in the commercialpackage insert.

Decitabine is4-amino-1-(2-deoxy-β-D-erythropentofuranosyl)-1,3,5-triazin-2(1H)-one:

DACOGEN® (decitabine for injection, Elsai, Inc., Woodcliff Lake, N.J.;DACOGEN® is a registered trademark of SuperGen, Inc., Dublin, Calif.) isindicated and approved by the US FDA for treatment of patients withmyelodysplastic syndromes (MDS) including previously treated anduntreated, de novo and secondary MDS of all French-American-Britishsubtypes (refractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts, refractory anemia with excessblasts in transformation, and chronic myelomonocytic leukemia) andintermediate-1, intermediate-2, and high-risk international PrognosticScoring System groups. Full prescribing information for DACOGEN® isavailable in the commercial package insert.

Compound Administration

It has now been discovered that the administration of an NAE inhibitoror a pharmaceutically acceptable salt thereof and a hypomethylatingagent or a pharmaceutically acceptable salt thereof can provide asynergistic effect.

The NAE inhibitor or a pharmaceutically acceptable salt thereof (NAEI)can be administered in combination with the hypomethylating agent or apharmaceutically acceptable salt thereof (HMA) in a single dosage formor as a separate dosage form. When administered as a separate dosageform, the hypomethylating agent or a pharmaceutically acceptable saltthereof can be administered prior to, at the same time as, or followingadministration of the NAE inhibitor or a pharmaceutically acceptablesalt thereof. As used herein, the administration in “combination” ofNAEI and HMA refers not only to simultaneous or sequentialadministration of the two agents, but also to the administration of bothcompounds during a single treatment cycle, as understood by one skilledin the art.

In some embodiments, the present disclosure relates to treating cancerin a patient by administering to the patient an NAE inhibitor or apharmaceutically acceptable salt thereof (NAEI) and a hypomethylatingagent or a pharmaceutically acceptable salt thereof (HMA) according to a28-day cycle as follows: administer NAEI on Days 1, 4, 8 and 11; andadminister HMA on Days 1, 2, 3, 4, 5, 8 and 9. Optionally, the firstcycle is 35 days with administration of NAEI on Days 1, 4, 11 and 15 andadministration of HMA on Days 8, 9, 10, 11, 12, 15 and 16, withsubsequent cycles of 28 days as described in the preceding sentence.

In some embodiments, the present disclosure-relates to treating cancerin a patient by administering to the patient an NAE inhibitor or apharmaceutically acceptable salt thereof (NAEI) and a hypomethylatingagent or a pharmaceutically acceptable salt thereof (HMA) according to a28-day cycle as follows: administer NAEI on Days 1, 3, and 5; andadminister HMA on Days 1, 2, 3, 4, 5, 8 and 9. Optionally, the firstcycle is 35 days with administration of NAEI on Days 1, 3, and 5 andadministration of HMA on Days 8, 9, 10, 11, 12, 15 and 16, withsubsequent cycles of 28 days as described in the preceding sentence.

In various embodiments, the NAEI may be((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate (“MLN4924”) or((1S,2S,4R)-4-[(6-{(1R,2S)-5-chloro-2-methoxy-2,3-dihydro-1H-inden-1-yl]amino}pyrimidin-4-yl)oxy)-2-hydroxycyclopentyl)methylsulfamate (“I-216”). In at least one embodiment, the NAEI is MLN4924. Inat least one embodiment, the NAEI is 1-216.

In various embodiments, the HMA may be azacitidine or decitabine. In atleast one embodiment, the HMA is azacitidine. In at least oneembodiment, the HMA is decitabine.

In various embodiments, MLN4924 is administered in combination withazacitidine. In various embodiments, MLN4924 is administered incombination with decitabine. In various embodiments, I-216 isadministered in combination with azacitidine. In various embodiments,I-216 is administered in combination with decitabine.

In various embodiments, the NAEI Is administered at a dose of about 20mg/m², 30 mg/m², 40 mg/m², 45 mg/m², 50 mg/m², 60 mg/m² or 75 mg/m². Invarious embodiments, the HMA is administered at a dose of about 75mg/m².

In various embodiments, the NAEI is administered intravenously. Invarious embodiments, the NAEI is administered orally. In variousembodiments, the NAEI Is administered subcutaneously. In variousembodiments, the HMA is administered intravenously or subcutaneously.

In some embodiments, the present disclosure relates to treating cancerin a patient by administering to the patient an NAEI and ahypomethylating agent HMA according to a 28-day cycle as follows:administer the NAEI on Days 1, 4, 8 and 11; and administer HMA on Days1, 2, 3, 4, 5, 8 and 9; wherein the NAEI is MLN4924 and HMA isazacitidine; wherein MLN4924 is administered intravenously at a dose ofabout 20 mg/m², 30 mg/m, 40 mg/m², 45 mg/m², 60 mg/m² or 75 mg/m²;wherein azacitidine is administered at a dose of about 75 mg/m²; andwherein the cancer is a hematologic malignancy. In various embodiments,the hematologic malignancy is acute myeloid leukemia (AML) ormyelodysplastic syndromes (MDS). In various embodiments, the hematologicmalignancy is AML. In various embodiments, the hematologic malignancy isMDS.

In some embodiments, the present disclosure relates to treating cancerin a patient by administering to the patient an NAEI and ahypomethylating agent HMA according to a 28-day cycle as follows:administer the NAEI on Days 1, 3, and 5; and administer HMA on Days 1,2, 3, 4, 5, 8 and 9; wherein the NAEI is MLN4924 and HMA is azacitidine;wherein MLN4924 is administered intravenously at a dose of about 20mg/m², 30 mg/m², 40 mg/m², 45 mg/m², 50 mg/m², 60 mg/ma or 75 mg/m²;wherein azacitidine is administered at a dose of about 75 mg/m²; andwherein the cancer is a hematologic malignancy. In various embodiments,the hematologic malignancy is acute myelold leukemia (AML) ormyelodysplastic syndromes (MDS). In various embodiments, the hematologicmalignancy is AML. In various embodiments, the hematologic malignancy isMDS.

Therapeutic Substance; Pharmaceutical Compositions.

The therapeutic substance can be a pharmaceutically acceptable salt. Insome embodiments, such salts are derived from inorganic or organic acidsor bases. For reviews of suitable salts, see, e.g., Berge et al., J.Pharm. Sci., 1977, 66, 1-19 and Remington: The Science and Practice ofPharmacy, 20th Ed., A. Gennaro (ed.), Lippincott Williams & Wilkins(2000).

Examples of suitable acid addition salts include acetate, adipate,alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate,citrate, camphorate, camphor sulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, lucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.

Examples of suitable base addition salts include ammonium salts; alkalimetal salts, such as sodium and potassium salts; alkaline earth metalsalts, such as calcium and magnesium salts; salts with organic bases,such as dicyclohexylamine salts, N-methyl-D-glucamine; and salts withamino acids such as arginine, lysine, and the like.

For example, Berge lists the following FDA-approved commerciallymarketed salts: anions acetate, besylate (benzenesulfonate), benzoate,bicarbonate, bitartrate, bromide, calcium edetate(ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate,chloride, citrate, dihydrochloride, edetate(ethylenediaminetetraacetate), edisylate (1,2-ethanedisulfonate),estolate (lauryl sulfate), esylate (ethanesulfonate), fumarate,gluceptate (glucoheptonate), gluconate, glutamate, glycollylarsanilate(glycollamidophenylarsonate), hexylresorcinate, hydrabamine(N,N′-di(dehydro-abietyl)ethylenediamine), hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isethionate (2-hydroxyethanesulfonate),lactate, lactobionate, malate, maleate, mandelate, mesylate(methanesulfonate), methylbromide, methylnitrate, methylsulfate, mucate,napsylate (2-naphthalenesulfonate), nitrate, pamoate (embonate),pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate(8-chlorotheophyllinate) and triethlodide; organic cations benzathine(N,N′-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine; andmetallic cations aluminum, calcium, lithium, magnesium, potassium,sodium and zinc.

Berge additionally lists the following non-FDA-approved commerciallymarketed (outside the United States) salts: anions adipate, alginate,aminosalicylate, anhydromethylenecitrate, arecoline, aspartate,bisulfate, butylbromide, camphorate, digluconate, dihydrobromide,disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydrolodide,methylenebls(salicylate), napadisylate (1,5-naphthalenedisulfonate),oxalate, pectinate, persulfate, phenylethylbarbiturate, picrate,propionate, thiocyanate, tosylate and undecanoate; organic cationsbenethamine (N-benzylphenethylamine), clemizole(1-p-chlorobenzyl-2-pyrrolildine-1′-ylmethylbenzimidazole),diethylamine, piperazine and tromethamine(tris(hydroxymethyl)aminomethane); and metallic cations barium andbismuth.

As used herein, “pharmaceutically acceptable carrier” refers to amaterial that is compatible with a recipient subject (a mammal, forInstance a human) and is suitable for delivering an active agent to thetarget site without terminating the activity of the agent. The toxicityor adverse effects, if any, associated with the carrier are, forexample, commensurate with a reasonable risk/benefit ratio for theintended use of the active agent.

The pharmaceutical compositions for use in the methods of the presentdisclosure can be manufactured by methods such as conventionalgranulating, mixing, dissolving, encapsulating, lyophilizing, oremulsifying processes, among others. Compositions can be produced invarious forms, including granules, precipitates, or particulates,powders, including freeze dried, rotary dried or spray dried powders,amorphous powders, tablets, capsules, syrup, suppositories, injections,emulsions, elixirs, suspensions or solutions. Formulations can containstabilizers, pH modifiers, surfactants, solubilizing agents,bioavailability modifiers and combinations of these.

Pharmaceutically acceptable carriers that can be used in thesecompositions include ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates or carbonates, glycine, sorbic acid, potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

These pharmaceutical compositions are formulated for pharmaceuticaladministration to a mammal, such as a human being. Such compositions canbe administered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally, or via an implanted reservoir.The term “parenteral” as used herein includes subcutaneous, intravenous,intraperitoneal, intramuscular, intra-articular, intra-synovial,Intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques. In some embodiments, the compositionsare administered orally, intravenously, or subcutaneously. In someembodiments, the compositions are administered orally. In someembodiments, the compositions are administered intravenously. Theseformulations can be designed to be short-acting, fast-releasing, orlong-acting. Furthermore, the compositions can be administered in alocal rather than systemic means, such as administration (e.g., byinjection) at a tumor site.

Pharmaceutical formulations can be prepared as liquid suspensions orsolutions using a liquid, such as an oil, water, an alcohol, andcombinations of these. Solubilizing agents such as cyclodextrins can beincluded. Pharmaceutically suitable surfactants, suspending agents, oremulsifying agents, can be added for oral or parenteral administration.Suspensions can include oils, such as peanut oil, sesame oil, cottonseedoil, corn oil and olive oil. Suspension preparations can also containesters of fatty acids such as ethyl oleate, Isopropyl myristate, fattyacid glycerides and acetylated fatty acid glycerides. Suspensionformulations can include alcohols, such as ethanol, isopropyl alcohol,hexadecyl alcohol, glycerol and propylene glycol; ethers, such aspoly(ethyleneglycol); petroleum hydrocarbons such as mineral oil andpetrolatum; and water.

Sterile injectable forms of these pharmaceutical compositions can beaqueous or oleaginous suspensions. These suspensions can be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationcan also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the Illustrative vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic add and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, for instance in theirpolyoxyethylated versions. These oil solutions or suspensions can alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsIncluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms can also be used for thepurposes of formulation. Compounds can be formulated for parenteraladministration by Injection such as by bolus injection or continuousinfusion. A unit dosage form for injection can be in ampoules or inmulti-dose containers.

For example, in various embodiments of the present disclosure, the NAEIis MLN4924 Injection Drug Product (“MLN4924-IDP”). MLN4929-IDP isformulated with the following excipients: citric acid; sodium hydroxide;Cyclodextrin Sulfobutylethers, Sodium Salts (Captisol®); and water forinjection. In at least one embodiment, MLN4929-IDP consists of 10 mg/mLMLN4924 (as free base) In a solution containing 50 mM citrate buffer and100 mg/mL sulfobutylether β-cyclodextrin, pH 3.3.

MLN4924-IDP has experienced stability problems when diluted in saline.MLN4924-IDP can be used for the duration of the retest period indicatedon the Certificate of Analysis. In practice, MLN4924-IDP has been storedrefrigerated at 5° C.±3° C. Each Type I glass vial nominally contains 5mL of compounded sterile solution, sealed with a Teflon®-coated butylrubber stopper and oversealed with an aluminum seal with a plasticFlip-Off® cap.

In various embodiments of the present disclosure, the HMA isazacitidine. Azacitidine is commercially available VIDAZA® (azacitidinefor injection), which is supplied as lyophilized powder in 100-mgsingle-use vials. Refer to the VIDAZA® package Insert for additionalinformation.

These pharmaceutical compositions can be orally administered in anyorally acceptable dosage form including capsules, tablets, aqueoussuspensions or solutions. When aqueous suspensions are required for oraluse, the active ingredient can be combined with emulsifying andsuspending agents. If desired, certain sweetening, flavoring or coloringagents can also be added. For oral administration in a capsule form,useful diluents include lactose and dried cornstarch. In the case oftablets for oral use, carriers that are commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. Coatings may be used for a variety of purposes,e.g., to mask taste, to affect the site of dissolution or absorption, orto prolong drug action. Coatings can be applied to a tablet or togranulated particles for use in a capsule.

Alternatively, these pharmaceutical compositions can be administered inthe form of suppositories for rectal administration. These can beprepared by mixing the agent with a suitable non-irritating excipientwhich is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum to release the drug. Such materialsinclude cocoa butter, beeswax and polyethylene glycols.

These pharmaceutical compositions can also be administered topically,for Instance when the target of treatment includes areas or organsreadily accessible by topical application, including diseases of theeye, the skin, or the lower intestinal tract. Suitable topicalformulations are readily prepared for each of these areas or organs.

Topical application for the lower intestinal tract may be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches can also be used. For topicalapplications, the pharmaceutical compositions can be formulated in asuitable ointment containing the active component suspended or dissolvedin one or more carriers. Carriers for topical administration of thecompounds of the present disclosure include mineral oil, liquidpetrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, thepharmaceutical compositions can be formulated in a suitable lotion orcream containing the active component(s) suspended or dissolved in atleast one pharmaceutically acceptable carrier. Suitable carriers includemineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions can be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or, forinstance, as solutions in Isotonic, pH adjusted sterile saline, eitherwith our without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions canbe formulated in an ointment such as petrolatum.

The pharmaceutical compositions can also be administered by nasalaerosol or Inhalation. Such compositions can be prepared according totechniques known in the art of pharmaceutical formulation and can beprepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other conventional solubilizing or dispersingagents.

The methods disclosed herein can be used to treat diseases, disorders,and conditions in which Inhibition of NAE enzyme activity is detrimentalto survival and/or expansion of diseased cells or tissue (e.g., cellsare sensitive to NAE inhibition; inhibition of NAE activity disruptsdisease mechanisms; reduction of NAE activity stabilizes protein whichare Inhibitors of disease mechanisms; reduction of NAE activity resultsin inhibition of proteins which are activators of disease mechanisms).The diseases, disorders and conditions can also include those whichrequire effective cullin and/or ubiquitination activity, which activitycan be regulated by diminishing NAE enzyme activity.

For example, the methods disclosed herein can be useful in treatment ofdisorders involving cellular proliferation, including disorders whichrequire an effective cullin-dependent ubiquitination and proteolysispathway (e.g., the ubiquitin proteasome pathway) for maintenance and/orprogression of the disease state. The methods of the present disclosurecan be useful in treatment of disorders mediated via proteins (e.g.,NFκB activation, p27^(Kip) activation, p21^(wAF/CP1) activation, p53activation) which are regulated by NAE activity. Representativedisorders include proliferative disorders, most notably cancers andinflammatory disorders (e.g., rheumatoid arthritis, inflammatory boweldisease, asthma, chronic obstructive pulmonary disease (COPD),osteoarthritis, dermatosis (e.g., atopic dermatitis, psoriasis),vascular proliferative disorders (e.g., atherosclerosis, restenosis)autoimmune diseases (e.g., multiple sclerosis, tissue and organrejection)); as well as inflammation associated with infection (e.g.,immune responses), neurodegenerative disorders (e.g., Alzheimer'sdisease, Parkinson's disease, motor neuron disease, neuropathic pain,triplet repeat disorders, astrocytoma, and neurodegeneration as resultof alcoholic liver disease), ischemic injury (e.g., stroke), andcachexia (e.g., accelerated muscle protein breakdown that accompaniesvarious physiological and pathological states, (e.g., nerve injury,fasting, fever, acidosis, HIV infection, cancer affliction, and certainendocrinopathies)).

The methods disclosed herein can be useful, for Instance, for thetreatment of cancer. As used herein, the term “cancer” refers to acellular disorder characterized by uncontrolled or disregulated cellproliferation, decreased cellular differentiation, inappropriate abilityto invade surrounding tissue, and/or ability to establish new growth atectopic sites. The term “cancer” includes solid tumors and bloodbornetumors. The term “cancer” encompasses diseases of skin, tissues, organs,bone, cartilage, blood, and vessels. The term “cancer” furtherencompasses primary and metastatic cancers.

In some embodiments, the cancer is a solid tumor. Examples of solidtumors that can be treated by the methods of the present disclosureinclude pancreatic cancer; bladder cancer; colorectal cancer; breastcancer, Including metastatic breast cancer; prostate cancer, includingandrogen-dependent and androgen-independent prostate cancer; renalcancer, including, e.g., metastatic renal cell carcinoma; hepatocellularcancer; lung cancer, including, e.g., small cell lung cancer (SCLC),non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC),and adenocarcinoma of the lung; ovarian cancer, including, e.g.,progressive epithelial or primary peritoneal cancer; cervical cancer;gastric cancer; esophageal cancer; head and neck cancer, including,e.g., squamous cell carcinoma of the head and neck; melanoma;neuroendocrine cancer, including metastatic neuroendocrine tumors; braintumors, including, e.g., glioma, anaplastic oligodendroglioma, adultglioblastoma multiforme, and adult anaplastic astrocytoma; bone cancer;and soft tissue sarcoma.

In some embodiments, the cancer is a hematologic malignancy. Examples ofhematologic malignancy include acute myelold leukemia (AML); chronicmyelogenous leukemia (CML), including accelerated CML and CML blastphase (CML-BP); acute lymphoblastic leukemia (ALL); chronic lymphocyticleukemia (CLL); Hodgkin's disease (HD); non-Hodgkin's lymphoma (NHL),including follicular lymphoma and mantle cell lymphoma; B-cell lymphoma;T-cell lymphoma; multiple myeloma (MM); Waldenstrom's macroglobulinemia;myelodysplastlc syndromes (MDS), including refractory anemia (RA),refractory anemia with ringed siderblasts (RARS), (refractory anemiawith excess blasts (RAEB), and RAEB in transformation (RAEB-T); andmyeloproliferative syndromes.

In some embodiments, a physician may diagnose a patient with a cancer aspredominantly one type. In some embodiments, a physician may diagnose apatient as having more than one type of cancer. In some embodiments, thediagnosis is predominantly one type of myelodysplastic syndromes. Insome embodiments, the diagnosis is more than one type of myelodysplasticsyndromes.

In some embodiments, methods of the present disclosure are used to treata patient having, or at risk of developing or experiencing, a recurrencein a tumor cancer, such as colorectal cancer, ovarian cancer, lungcancer, breast cancer, gastric cancer, prostate cancer and pancreaticcancer. In some embodiments, methods of the present disclosure are usedto treat a patient having, or at risk of developing or experiencing, arecurrence in a hematologic cancer, such as AML, CML, CML-BP, ALL, orCLL.

In order that this disclosure be more fully understood, the followingexamples are set forth. These examples are illustrative only and are notintended to limit the scope of the present disclosure in any way.

EXAMPLES 1. In Vitro Cell Viability Assays

The experimental protocol used Poly-D-lysine BloCoat™ Black/Clear 384plates (Becton Dickinson, Franklin Lakes, N.J.). The appropriate NAEinhibitor was dissolved in DMSO and delivered Into the wells using anEcho (Labcyte, Sunnyvale, Calif.) liquid handling system. HL60 and THP-1lines were obtained from ATCC (American Type Culture Collection,Manassas, Va.), while NB4 and OCI-M2 lines were obtained from DSMZ(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Brunswick,Germany). Each plate had a cell suspension from one of the lines addedto the wells. A portion of the wells were used as positive controls (nocompound was added), while another portion of the wells were used asnegative controls (no cells were added). The plates were incubated for72 hours, and then the cell viabilities were measured using an ATPlite(PerkinElmer, Waltham, Mass.) assay.

Statistical Analyses.

Normalization. The viability data was normalized separately for eachplate by scaling the data so that the median of the negative controlswas 0 and the median of the positive controls was 100. More formally,

$V_{i} = {100\frac{U_{i} - {{median}\left( U_{-} \right)}}{{{median}\left( U_{+} \right)} - {{median}\left( U_{-} \right)}}}$

where V_(i) is the normalized viability of the i^(th) well, U_(i) is theraw viability measurement, median (U⁻) is the median of the negativecontrols, and median(U₊) is the median of the positive controls. Afternormalization, the controls were discarded.

Response surface model and fitting. A response surface model was used todescribe the relationship between the normalized viability and the drugconcentrations. For a given plate, let

C=(C _(A) /I ₁)+(C _(B) /I ₂)

x=(C _(A) /I ₁)/C

E _(max) =E ₁ +E ₂ x+E ₃ x ² +E ₄ x ³

I=1+I ₃ x(1−x)

S=S ₁ +S ₂ x+S ₃ x ² +S ₄ x ³

V=100−E _(max)(1+(I/C)^(S))⁻¹+error

where E₁, E₂, E₃, E₄, I₁, I₂, I₃, S₁, S₂, S₃, and S₄ are parameters,C_(A) and C_(B) are the respective concentrations of drugs A and B, andV is the normalized viability measurement. It was assumed that the errorvalues were independent and identically distributed normal randomvariables. This model is an extension of the Hill equation (A. V. Hill,J. Physiol., 1910, 40, iv-vii), which is commonly used to model theeffect of a single drug. The data were fitted to this model using themaximum likelihood method with the statistical software program R RDevelopment Core Team (2008) (R: A language and environment forstatistical computing. R Foundation for Statistical Computing, Vienna,Austria. ISBN 3-900051-07-0, URL http://www.R-project.org).

Quality checks. Three types of quality checks were applied to theplates. First, it was checked that the variation of the positivecontrols and the mean of the negative controls were small. Next, it waschecked that the new data agreed with data from previous single drugexperiments. Finally, the residuals from the response surface fit wereanalyzed to ensure that the residual sum of squares was sufficientlysmall. All of these quality checks were based on numerical thresholds tomake pass/fail decisions, and the same thresholds were used for all ofthe plates in the experiment. If a plate failed any one of the qualitychecks, It was removed from the analysis.

Measuring in vitro synergy. The Combination index (M. C. Berenbaum, J.Theor. Biol., 1985, 114, 413-431) was used as a measure of drug synergy.The Combination Index is computed based on an isobologram, which is aslice of the dose response surface with constant viability. For thepresent analysis, the 50% isobologram, which is the dose contour thathas 50% viability, was used. The EC50_(A) and EC50_(B) are defined bethe respective doses of drugs A and B alone that have a viability of50%. For a point (D_(A), D_(B)) along the 50% Isobologram, theCombination Index is defined as (D_(A)/EC50_(A))+(D_(B)/EC50_(B)). Sincethe choice of (D_(A), D_(B)) can be arbitrary, the constraintD_(A)/D_(B)=EC50_(A)/EC50_(B) was used. If the Combination Index is lessthan 1, it Indicates that the isobologram curves inward, and that thedrug combination is synergistic. Conversely, if the Combination Index Isgreater than 1, the 50% isobologram curves outward, indicatingantagonism. In the more stringent analysis method applied according tothe present disclosure, Combination Index values within the range0.8-1.2 are considered additive. This rule prevents small deviationsfrom additivity from being classified as synergistic.

A two sided t-test for each condition was performed to determine if themean Combination index differed from 1. The Benjaminl-Hochberg method(Y. Benjamini and Y. Hochberg, J. R. Stat. Soc., Series B (Stat.Methodol.), 1995, 57 (1), 289-300) was used to adjust the resultingp-values for multiple hypothesis testing. An adjusted p-value below 0.05was considered to statically significant. In order for a combination tobe classified as synergistic, we required that three criteria be met:the mean Combination Index for the condition had to be less than 1, thedifference had to be statistically significant, and Combination Indexhad to be outside the range (0.8, 1.2). This third criterion preventedsmall deviations from additivity from being classified as synergistic.Combinations for which the p-value was above 0.05 or the CombinationIndex was within the range (0.8, 1.2) were classified as additive.

Cell viability assays were used to assess the combination effect invitro of each of two NAE inhibitors, MLN4924 and I-216, with each of twohypomethylating agents, azacitidine and decitabine, in four cell lines,HL60, OCIM2, NB4, and THP-1. FIG. 1 shows the Combination Index for allof the experiments that passed the quality checks among each testedcombination. The results are arranged by the condition. Table 1, below,lists the mean Combination Index, the adjusted p-value, and theconclusion for each determined combination. As Table 1 shows, all eightcombinations of NAE Inhibitor and hypomethylating agent demonstrated asynergistic effect in both the OCIM2 and NB4 cell lines. In the HL60line, both NAE inhibitors demonstrated synergy with decitabine andshowed an additive effect with azacitidine. In the THP-1 line, both NAEinhibitors demonstrated an additive effect with azacitidine. Due to thelack of single agent activity of decitabine in THP-1, a CombinationIndex cannot be calculated for the in vitro experiments with NAEinhibitors and decitabine in THP-1.

TABLE 1 Summary of the Combination Index values. Number of Mean NAEHypomethylating passing Combination Adjusted inhibitor agent Cell lineplates Index P-value Conclusion MLN4924 Decitabine HL60 6 0.45 6.2 ×10⁻⁴ Synergy I-216 Decitabine HL60 4 0.46 5.8 × 10⁻³ Synergy MLN4924Azacitidine HL60 5 1.03 1.1 × 10⁻¹ Additivity I-216 Azacitidine HL60 30.97 4.9 × 10⁻¹ Additivity MLN4924 Decitabine OCIM2 7 0.52 1.4 × 10⁻⁴Synergy I-216 Decitabine OCIM2 5 0.45 4.0 × 10⁻⁵ Synergy MLN4924Azacitidine OCIM2 5 0.44 4.8 × 10⁻⁶ Synergy I-216 Azacitidine OCIM2 30.41 2.7 × 10⁻³ Synergy MLN4924 Decitabine NB4 5 0.61 1.4 × 10⁻⁴ SynergyI-216 Decitabine NB4 4 0.61 2.7 × 10⁻³ Synergy MLN4924 Azacitidine NB4 40.52 5.8 × 10⁻³ Synergy I-216 Azacitidine NB4 4 0.56 5.3 × 10⁻³ SynergyMLN4924 Decitabine THP-1 4 NA NA NA I-216 Decitabine THP-1 4 NA NA NAMLN4924 Azacitidine THP-1 4 1.10 2.4 × 10⁻² Additivity I-216 AzacitidineTHP-1 4 1.11 2.7 × 10⁻² Additivity

FIG. 1 shows the Combination Index values for each plate, arranged bythe condition (i.e., a given drug combination applied to a given cellline). These results were summarized by computing the mean CombinationIndex for each condition, as shown in Table 1.

2. In Vivo Tumor Efficacy Models Subcutaneous Xenograft Models

Test subjects. HL-60 (2×10⁶) tumor cells in 100 μL phosphate bufferedsaline with Matrigel™ (BD Biosciences, Bedford, Mass.) were asepticallyinjected into the subcutaneous space in the right dorsal flank of femaleNcr nude mice (age 5-8 weeks, Charles River Laboratories, Wilmington,Mass.) using a 26-gauge needle. THP-1 (2.5×10⁶) or OCI-M2 (5×10⁶) tumorcells in 100 μL phosphate buffered saline with Matrigel™ wereaseptically injected into the subcutaneous space in the right dorsalflank of female CB.17 SCID mice (age 5-8 weeks, Charles RiverLaboratories) using a 26-gauge needle.

Beginning on day seven (7) after Inoculation, tumors were measured twiceweekly using a vernier caliper. Tumor volumes were calculated usingstandard procedures (0.5×(length×width²)). When the tumors reached avolume of approximately 200 mm³, mice were randomized into groups of 10and injected subcutaneously with compound inhibitor (200 μL) at variousdoses and schedules, with the first dosing day defined as Day 1. Allcontrol groups received vehicle alone. Tumor size and body weight weremeasured approximately twice a week for the duration of the study. Micewere euthanized when their tumor volume reached 10% of their bodyweight, or when the average tumor volume of a treatment or control groupreached approximately 2000 mm³. The dosing schedule for each study wasas follows: MLN4924 and azacitidine were dosed separately or co-dosed bysubcutaneous injection on Days 1, 4, 8, 11, 15 and 18 at the indicateddoses. Tumor growth continued to be monitored after the dosing period.Average tumor volume reported as a function of time is shown in FIGS.2-4.

Statistical Analyses of Synergy for Tumor Growth in SubcutaneousXenograft Models.

For the THP-1 and OCI-M2 models, measurements from day 0 to 21 wereanalyzed. For the HL60 model, measurements from day 0 to 14 were used,since several of the mice had tumors exceeding the allowed volume afterday 14. All tumor volumes had a value of 1 added to them before log₂₀transformation. These values were compared across treatment groups toassess whether the differences in the trends over time werestatistically significant. To compare pairs of treatment groups, thefollowing mixed-effects linear regression model was fit to the datausing the maximum likelihood method:

Y _(ijk) −Y _(i0k) =Y _(i0k)+treat_(i)+day_(j)+day_(j)²+(treat*day)_(ij)+(treat*day²)_(ij) +e _(ijk)

where Y_(ijk) is the log₁₀ tumor value at the j^(th) time point of thek^(th) animal in the i^(th) treatment, Y_(i0k) is the day 0 log₁₀ tumorvalue in the k^(th) animal in the i^(th) treatment, day_(j) was themedian-centered time point and was treated as a continuous variable, ande_(ijk) is the residual error. A spatial power law covariance matrix wasused to account for the repeated measurements on the same animal overtime. Interaction terms as well as day_(j) ² terms were removed if theywere not statistically significant.

A likelihood ratio test was used to assess whether a given pair oftreatment groups exhibited differences which were statisticallysignificant. The −2 log likelihood of the full model was compared to onewithout any treatment terms (reduced model) and the difference in thevalues was tested using a Chi-squared test. The degrees of freedom ofthe test were calculated as the difference between the degrees offreedom of the full model and that of the reduced model.

In addition to the statistical significance, a measure of the magnitudeof the effect for each treatment was found. The predicted differences inthe log tumor values (Y_(ijk)−Y_(i0k)) vs. time were taken from theabove model to calculate mean area under the curve (AUC) values for eachtreatment group. A dAUC value was then calculated as:

${dAUC} = {100\frac{{{mean}\left( {AUC}_{control} \right)} - {{mean}\left( {AUC}_{treatment} \right)}}{{{mean}\left( {AUC}_{control} \right)}}}$

For synergy analyses, the observed differences in the log tumor valueswere used to calculate AUC values for each animal. In instances when ananimal in a treatment group was removed from the study, the lastobserved tumor value was carried forward through all subsequent timepoints. To Improve the robustness of the synergy analysis, the followingprocedure was applied to the AUC values from each treatment group. Let xbe the set of AUC values for a given treatment group. A range ofinterest was defined:

(median(x)−5*MAD(x), median(x)+5*MAD(x)).

Here, MAD is the median absolute deviation of x. If any value in x felloutside this range, that value was replaced by the value at the closestboundary. The procedure was non-iterative, so the range was computedonly once for each treatment group.

The synergy score for the combination of treatments A and B was definedas

100*(mean(AUC_(AB))−mean(AUC_(A))−mean(AUC_(B))+mean(AUC_(ctl)))/mean(AUC_(ctl))

where AUC_(AB), AUC_(A), AUC_(B), and AUC_(ctl) are the AUC values foranimals in the combination group, the A group, the B group, and thecontrol group, respectively. The standard error of the synergy score wascomputed based on the variation in the AUC values among the animals. Atwo sided t-test was used to determine if the synergy score wassignificantly different from zero. If the P-value was below 0.05, andthe synergy score was less than zero, then the combination wasconsidered to be synergistic. If the P-value was above 0.05, then thecombination was considered to be additive.

Mouse xenograft models were used to assess the combination effect invivo of NAE inhibitor MLN4924 and hypomethylating agent azacitidine.FIGS. 2-4 show tumor volume as a function of time in three subcutaneousxenograft models following treatment with the vehicle as a single agent,MLN4924 as a single agent, azacitidine (“Aza”) as a single agent, andco-administration (s.c.) of MLN4924 and azacitidine on Days 1, 4, 8, 11,11, 15 and 18 at the Indicated doses.

In the HL-60 subcutaneous xenograft model (FIG. 2), MLN4924 andazacitidine as single agents had a marginal effect on tumor growth. Incontrast, co-dosing MLN4924 and azacitidine led to tumor regressions,with a statistical assessment of synergy.

In the THP-1 xenograft model (FIG. 3), azacitidine as single agent had amarginal effect on tumor growth whereas MLN4924 as a single agentinhibited tumor growth. In contrast, co-dosing MLN4924 and azacitidineled to tumor regressions. Despite the statistical assessment ofadditivity in this model, rather than synergy, the figure clearly showsa combination benefit: tumor growth inhibition (single agent) versustumor regression with the combination.

An additional demonstration of improved activity in THP-1 is the delayin tumor regrowth with the combination compared to each single agent.The additional benefit provided by the combination over the single agenttreatments was statistically significant, as shown in Table 3b (P-value<0.05).

In the OCI-M2 subcutaneous xenograft model (FIG. 4), MLN4924 andazacitidine as single agents inhibited tumor growth. In contrast,co-dosing MLN4924 and azacitidine led to tumor regressions with astatistical assessment of synergy.

Disseminated Xenograft Model

Test subjects. HL-60 (1×10⁷) tumor cells in 100 μL IMDM media wereinoculated in the lateral vein of female mice CB-17 SCID (age 8-10weeks, Charles River Laboratories, Wilmington, Mass.) using a 27-gaugeneedle. On day 20 post-inoculation, mice were randomized into groups of10. Starting on day 22, mice were dosed subcutaneously with vehicle, 180mg/kg MLN4924, 10 mg/kg azacitidine, or the combination of 180 mg/kgMLN4924 and 10 mg/kg azacitidine, using the same twice-weekly scheduleas described in the subcutaneous xenograft experiments (dosing on days22, 25, 29, 32, 36, 39). The mice were monitored at least twice weeklyfor body weight loss and signs of disease, including paresis orparalysis of hind limbs and emergence of palpable and internal solidtumors. The day on which an animal died or was sacrificed due to diseaseburden was recorded. Survival time is shown in FIG. 5.

Statistical Analysis of Synergy for Survival in Disseminated XenograftModel.

To determine synergy in the survival times, the mean survival times andcorresponding standard errors were computed for each treatment group.The survival synergy was defined as

mean(survival_(AB))−mean(survival_(A))−mean(survival_(B))+mean(survival_(ctl))

where survival_(AB), survival_(A), survival_(B), and survival_(ctl) arethe survival times for animals in the combination group, the A group,the B group, and the control group, respectively. The standard error forthe survival synergy was found by adding the standard error of each ofthe four terms in quadrature. A two sided Z-test was used to determineif the survival synergy was significantly different from zero. If theP-value was below 0.05, and the survival synergy was greater than zero,then the combination was considered to be synergistic, if the P-valuewas above 0.05, then the combination was considered to be additive.

FIG. 5 shows survival as a function of time in a disseminated xenograftmodel in which HL-60 cells were inoculated by intravenous injection, andmice were treated with vehicle, with MLN4924 as a single agent,azacitidine as a single agent, and co-administration of s.c. MLN4924 andazacitidine beginning on Day 22 post-inoculation at the indicated doses,using the same twice-weekly schedule as in the experiments in FIGS. 2-4.In the HL-60 disseminated model (FIG. 5), MLN4924 and azacitidine assingle agents both extended mean survival time compared to the controlgroup (8.4 day extension for MLN4924 and 21.1 day extension forazacitidine). The combination of MLN4924 and azacitidine extended meansurvival time by 36.7 days, which is 7.2 days longer than would beexpected from an additive combination (FIG. 5). The survival synergy wasstatistically significant.

TABLE 2A Synergy assessment for HL60 subcutaneous xenograft tumors.Synergy Sunergy score Treatment score standard error P-Value AssessmentMLN4924 180 mg/kg + azacitidine 15 mg/kg −54.8 17.6 0.005 Synergy

TABLE 2B Pairwise comparison of treatment groups for HL60 subcutaneousxenograft tumors. P-Value for the Reference Treated dAUC difference ineffects MLN4924 180 mg/kg MLN4924 180 mg/kg + azacitidine 15 mg/kg 118.3<0.001 azacitidine 15 mg/kg MLN4924 180 mg/kg + azacitidine 15 mg/kg112.9 <0.001

TABLE 3A Synergy assessment for THP-1 subcutaneous xenograft tumors.Synergy Synergy score Treatment score standard error P-Value AssessMLN4924 180 mg/kg + azacitidine 10 mg/kg −40.5 21.4 0.078 Additive

TABLE 3B Pairwise comparison of treatment groups for THP-1 subcutaneousxenograft tumors. P-Value for the Reference Treated dAUC difference ineffects MLN4924 180 mg/kg MLN4924 180 mg/kg + azacitidine 10 mg/kg 882.2<0.001 azacitidine 10 mg/kg MLN4924 180 mg/kg + azacitidine 10 mg/kg221.1 <0.001

TABLE 4A Synergy assessment for THP-1 subcutaneous xenograft tumors.Synergy score Treatment Synergy score standard error P-Value AssessmentMLN4924 180 mg/kg + azacitidine 5 mg/kg −52.1 15.1 0.002 Synergy

TABLE 4B Pairwise comparison of treatment groups for OCI-M2 subcutaneousxenograft tumors. P-Value for the Reference Treated dAUC difference ineffects MLN4924 180 mg/kg MLN4924 180 mg/kg + azacitidine 5 mg/kg 545.5<0.001 azacitidine 5 mg/kg MLN4924 180 mg/kg + azacitidine 5 mg/kg 767.6<0.001

TABLE 5A Mean survival times for mice with HL-60 disseminated xenograftmodel Mean Standard error survival of mean survival Treatment time(days) time (days) Vehicle 48.1 1.0 MLN4924 180 mg/kg 56.5 0.7azacitidine 10 mg/kg 69.2 2.1 MLN4924 180 mg/kg + 84.8 2.4 azacitidine10 mg/kg

TABLE 5B Survival time synergy assessment for HL-60 disseminatedxenograft model. Survival Survival synergy Treatment synergy (days)standard error (days) P-Value Assess MLN4924 180 mg/kg + azacitidine 10mg/kg 7.2 3.4 0.036 Synergy

Prophetic Drug Administration Example.

Prior to use, MLN4924-IDP vials are warmed to ambient conditions (15° C.to 30° C.) by placing them at room temperature. Accelerated warmingmethods, such as a water bath, was not, and must not be, used.MLN4924-IDP is stable at room temperature for 8 hours prior to dilution.

Each MLN4924-IDP vial contains nominally 5 mt (50 mg MLN4924 as freebase). Using sterile technique, the appropriate volume of drug Iswithdrawn from vial(s) and injected into a 250 mL IV bag containing a 5%dextrose solution, which is then gently inverted repeatedly to mix. Theprepared MLN4924-IDP IV bag must be used within 6 hours if stored atroom temperature. Alternatively, the prepared IV bag is chemicallystable and can be stored for up to 24 hours at 5° C.±3° C. After 24hours of storage at 5° C.±3° C., the prepared IV bag must be used within6 hours upon coming to room temperature. The vial must not be shaken atany time during dose preparation.

Instructions for the preparation, reconstitution, and dispensation ofazacitidine are provided in the azacitidine (VIDAZA®) package Insert

The amount of MLN4924 and azacitidine administered is based on bodysurface area (BSA). BSA is calculated using a standard nomogram on Cycle1, Day 1, and at subsequent visits if the patient experiences a >5%change in body weight from the weight used for the most recent BSAcalculation.

Patients receive MLN4924 diluted with 5% dextrose in a 250-mL IV bag viaa 60-minute infusion. MLN4924 should be administered through central orperipheral venous access. The infusion can be slowed or stopped andrestarted for any associated infusion reactions. The total infusion timemust not exceed six hours from the time of reconstitution.

The entire content of the MLN4924 IV bag will be infused at a constantrate over 1 hour. To ensure that all the MLN4924 enters the body, theinfusion line will be flushed with 5% dextrose immediately afteradministration.

Instructions for the administration of azacitidine are provided in theazacitidine (VIDAZA®) package Insert.

Although DLTs can occur at any point during treatment, only DLTsoccurring during Cycle 1 of treatment will necessarily influencedecisions regarding dose escalation, expansion of a dose level, orevaluation of Intermediate dose levels. Patients are monitored throughall cycles of therapy for treatment-related toxicities.

The duration of cycles will be 28 days. Azacitidine will be administeredin a 5-on/2-off/2-on schedule, i.e., on Days 1, 2, 3, 4, 5, 8, and 9.MLN4924 will be administered on Days 1, 3, and 5. Patients will receiveboth agents on Days 1, 3 and 5. MLN4924 can be administered at a dose of20 mg/m², 30 mg/m², 40 mg/m², 45 mg/m², 50 mg/m², 60 mg/m², or 75 mg/m².Azacitidine will be administered either IV or SC at a dose of 75 mg/m².

In an optional embodiment, MLN4924 will be administered on Days 1, 8,and 15.

Optionally, the duration of cycles will be 28 days, with the exceptionof Cycle 1, where a 7-day lead-in will be incorporated where noazacitidine will be administered, such that Cycle 1 will last a total of35 days. According to this schedule, azacitidine will be administered ina 5-on/2-off/2-on schedule; on Days 8 to 12 and Days 15 and 16 In Cycle1, and on Days 1, 2, 3, 4,5, 8, and 9 for all subsequent cycles.According to this schedule, MLN4924 will be administered on Days 1, 3,and 5.

In an another optional embodiment, MLN4924 will be administered on Days1, 4, 11 and 15 for Cycle 1 only, giving one 35 day cycle; in allsubsequent cycles, MLN4924 will be administered on Days 1, 3, and 5,each cycle lasting 28 days. According to this optional schedule,patients will receive both agents on Days 11 and 15 of Cycle 1 and onDays 1, 3, and 5 of subsequent cycles. MLN4924 will be administered at adose of 20 mg/m², 30 mg/m², 40 mg/m², 45 mg/m², 50 mg/m², 60 mg/m², or75 mg/m². Azacitidine will be administered either IV or SC (physician'schoice) at a dose of 75 mg/m².

Patients will receive azacitidine as either an IV or SC injection (seeazacitidine [VIDAZA®]package insert for details on administration). Ondays where both MLN4924 and azacitidine are to be administered, infusionof MLN4924 will commence at a time ranging from 15 to 60 minutes aftercompletion of administration of azacitidine. An assessment of vitalsigns will be made pre azacitidine dose, pre MLN4924 dose, and postMLN4924 dose on these days.

What is claimed is:
 1. A method of treating cancer comprising,administering to a patient in need of such treatment a therapeuticallyeffective total amount of an NAE inhibitor or a pharmaceuticallyacceptable salt thereof, and a hypomethylating agent or apharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the hypomethylating agent is azacitidine or decitabine, or apharmaceutically acceptable salt thereof.
 3. The method of claim 2,wherein the hypomethylating agent is azacitidine or a pharmaceuticallyacceptable salt thereof.
 4. The method of claim 2, wherein thehypomethylating agent is decitabine or a pharmaceutically acceptablesalt thereof.
 5. The method of claim 1, wherein the NAE inhibitor is((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate or{(1S,2S,4R)-4-(6-{[(1R,2S)-5-chloro-2-methoxy-2,3-dihydro-1H-inden-1-yl]amino}pyrimidin-4-yl)oxy]-2-hydroxycyclopentyl)methylsulfamate, or a pharmaceutically acceptable salt thereof.
 6. The methodof claim 5, wherein the NAE inhibitor is((1S,2S,4R)-4-(4-((15)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methysulfamate or a pharmaceutically acceptable salt thereof.
 7. The methodof claim 5, wherein the NAE inhibitor is{(1S,2S,4R)-4-[(6-{[(1R,2S)-5-chloro-2-methoxy-2,3-dihydro-1H-inden-1-yl]amino}pyrimidin-4-yl)oxy]-2-hydroxycyclopentyl)methylsulfamate, or a pharmaceutically acceptable salt thereof.
 8. The methodof claim 6, wherein((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate or pharmaceutically acceptable salt thereof is administered onDays 1, 3, and 5 of a 28-day cycle.
 9. The method of claim 8, wherein((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate or pharmaceutically acceptable salt thereof is administered ata dose of about 20 mg/m².
 10. The method of claim 8, wherein((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-4H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate or pharmaceutically acceptable salt thereof is administered ata dose of about 30 mg/m².
 11. The method of claim 8, wherein((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate or pharmaceutically acceptable salt thereof is administered ata dose of about 40 mg/m².
 12. The method of claim 8, wherein((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate or pharmaceutically acceptable salt thereof is administered ata dose of about 50 mg/m².
 13. The method of claim 8, wherein((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate or pharmaceutically acceptable salt thereof is administeredintravenously.
 14. The method of claim 8, wherein((1S,2S,4R)-4-(4-((1S)-2,3-dihydro-1H-inden-1-ylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methylsulfamate or pharmaceutically acceptable salt thereof is administeredsubcutaneously.
 15. The method of any one of claim 1, wherein thehypomethylating agent is azacitidine or a pharmaceutically acceptablesalt thereof and is administered on Days 1, 2, 3, 4, 5, 8 and 9 of a28-day cycle.
 16. The method of claim 15, wherein the azacitidine orpharmaceutically acceptable salt thereof is administered at a dose ofabout 75 mg/m².
 17. The method of claim 15, wherein the azacitidine orpharmaceutically acceptable salt thereof is administered subcutaneously.18. The method of claim 15, wherein the azacitidine or pharmaceuticallyacceptable salt thereof is administered intravenously.
 19. The method ofclaim 1, wherein the NAE inhibitor or pharmaceutically acceptable saltthereof is administered in combination with the hypomethylating agent orpharmaceutically acceptable salt thereof in a single dosage form. 20.The method of claim 1, wherein the NAE Inhibitor or pharmaceuticallyacceptable salt thereof is administered in combination with thehypomethylating agent or pharmaceutically acceptable salt thereof inseparate dosage forms.
 21. The method of claim 1, wherein the cancer isa hematologic malignancy.
 22. The method of claim 21, wherein the canceris acute myeloid leukemia (AML).
 23. The method of claim 21, wherein thecancer is myelodysplastic syndromes (MDS).
 24. The method of claim 23,wherein the myelodysplastic syndromes (MDS) are diagnosed as any ofrefractory anemia (RA), refractory anemia with ringed siderblasts(RARS), (refractory anemia with excess blasts (RAEB), and RAEB intransformation (RAEB-T).
 25. The method of claim 24, wherein thediagnosis is predominantly one type of myelodysplastic syndromes. 26.The method of claim 24, wherein the diagnosis is more than one type ofmyelodysplastic syndromes.
 27. The method of claim 21, wherein thecancer is diagnosed as any of chronic myelogenous leukemia (CML), acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL),Hodgkin's disease (HD), non-Hodgkin's lymphoma (NHL), T-cell lymphoma,multiple myeloma (MM), Waldenstrom's macroglobulinemia, myelodysplasticsyndromes (MDS), and myeloproliferative syndromes.
 28. The method ofclaim 27, wherein the diagnosis is predominantly one type of cancer. 29.The method of claim 27, wherein the diagnosis is more than one type ofcancer.
 30. A kit for treating cancer in a subject in recognized needthereof comprising: at least one medicament comprising at least one doseof an NAE Inhibitor or a pharmaceutically acceptable salt thereof, andat least one medicament comprising at least one dose of ahypomethylating agent or a pharmaceutically acceptable salt thereof;said kit for treating cancer further comprising dosing instructions foradministering the medicaments for treatment of the subject in recognizedneed thereof.